Recent rapid developments in genome and meta-genome sequencing has resulted in a large number of genes which represent a wealth of potentially very interesting proteins. Problems to express these genes at a significant level hamper the exploration of the functionality of the proteins encoded by those genes and as a consequence prevent the potential exploitation of such proteins in an economical feasible way. Since in many cases the discovered genes originate from organism which are less suitable for large scale production or which are rather inaccessible to the present genetic engineering tools, it is highly desired to use well established production hosts for which gene transfer systems and well developed genetic engineering tools are available. In particular eukaryotes such as filamentous fungi and yeasts are widely used as cell factories in the production of proteins, in particular the production of extracellular proteins. Because of a long tradition of utilization several of these species are generally regarded as safe (GRAS), which makes them very interesting for manufacture of products for human use. However, despite substantial improvements, the production levels obtained for heterologous genes are often much lower than observed for homologous genes. Often there is no expression of protein at all.
Various techniques exist to increase levels of protein production. These include application of strong promoters, increase of copy number, optimal Kozak sequence, mRNA stabilizing elements, optimized codon usage (WO2008/000632) and gene. These strategies however generally do not guarantee that proteins can be produced at detectable levels. To date the most successful approach for producing heterologous proteins is to express them as translational fusion with an efficiently secreted homologous protein. Nevertheless production levels still lag significantly behind and in many cases expression levels are problematically low. In general low expression in the fermentation leads to lower yields in the recovery. Even if expression is optimized, the final mature protein product may still result in very low production yields due to large losses in the downstream processing. This may be the case when the expressed protein remains associated with the biomass. This results in high losses or alternatively requires use of costly, and sometimes undesirable use of detergents to solubilise the proteins.